Carotenoids are a class of biochemicals consisting of the carotenes. Xanthophylls are a related class of biochemicals and are the oxygenated derivatives of carotenoids. The prefix xe2x80x9capoxe2x80x9d with an associated number or locant, signifies that all of the carotene molecule beyond that indicated part has been replaced by hydrogen atoms. Because these molecules contain a long series of conjugated double bonds, they often are highly colored, most often with red-to-yellow pigmentation. The most familiar carotenoid is xcex2-carotene, which is commonly found in carrots, in sweet potatoes and other yellow vegetables, and in green, leafy vegetables such as spinach and kale. The carotenoids, xanthophylls and apo-carotenoids have value as both colorants and nutritional dietary supplements.
xcex2-carotene is used as a food additive both as a colorant and a dietary supplement (Bauerfeind, J. C., et al. (1971) xe2x80x9cUse of carotenoids.xe2x80x9d In: Carotenoids. O. Isler et al. (eds.), Halsted Press, New York, pp.743-770.). In these applications, the usual chemical form of xcex2carotene is the all-trans configuration, which is the isomer found in root vegetables and in the synthetic material produced by the principal manufacturing technologies.
xcex2-carotene has been indicated to play a role in the prevention of cancer (Hennekens, et al., (1986) Cancer 58: 1837-1841; Krinsky, N. I. (1971) xe2x80x9cFunction.xe2x80x9d In: Carotenoids, O. Isler et al. (eds.), Halsted Press, New York, pp. 669-716.; Krinsky, N. I. (1988) Clin. Nutr. 7: 107-112; Mathews-Roth, M. M., and Krinsky, N. I. (1987) Photochem. and Photobiol. 40(4): 507-509; Krinsky et al., (1982) J. Natl. Cancer Institute 69: 205-210; Menkes, M., et al. (1986) New England J. of Med. 315: 1250-1254). Carotenoids such as xcex2-carotene, and xanthophylls such astaxanthin, have also been shown to play central roles in the metabolism of the eye""s macula and retina and in maintaining healthy vision (Handelman, G. J., and Dratz, E. A. (1986) xe2x80x9cThe role of antioxidants in the retina and retinal pigment epithelium and the nature of prooxidant-induced damage.xe2x80x9d Adv. in Free Radical Biology and Medicine, Vol. 2, pp. 1-89). Most Americans do not receive what is regarded by some as an optimal minimum dosage of approximately 75,000 I. U. (International Units) per day (Taylor, R. F., and Little. A. D. (1990) xe2x80x9cCarotenoids: products, applications, and markets.xe2x80x9d SPECTRUM Food Industry, pp. 1-11).
The xanthophyll pigment astaxanthin is widely distributed in nature and is the predominant pigment in shrimp, crab, lobster, and salmonids. Additionally, it produces the red coloration of some birds such as flamingos and the scarlet ibis (Weedon, B. C. L. (1971) xe2x80x9cOccurrence.xe2x80x9d In: Carotenoids, O. Isler et al. (eds.), Halsted Press, New York, pp. 29-60.). There is evidence that xanthophylls function as chemo-protectives. In addition, other xanthophylls, such as adonirubin and astaxanthin, may also act as nutraceuticals that prevent carcinogenesis through anti-oxidative, anti-free radical, or other mechanisms. The beneficial nutraceutical functions of the carotenes and xanthophylls extend to the prevention of heart attacks and strokes).
One of the most important uses of the xanthophylls is in animal feed. Astaxanthin provides the distinctive coloration for many multicellular organisms and generally must be obtained from a dietary source. When fish such as salmon, rainbow trout, red sea bream, or yellowtail are aquacultured, this pigment must be included as a dietary supplement in order to produce the coloration necessary for effective marketing (Committee on Animal Nutrition, National Research Council (1983) Nutrient Requirements of Warmwater Fishes and Shellfishes. National Academy of Science, pp. 55-57; Foss, P., et al., (1984) Aquaculture 41: 213-226; Foss, P., et al., (1987) Aquaculture 65: 293-305; Meyers, S. P., and Chen., H-M. (1982) xe2x80x9cAstaxanthin and its role in fish culture,xe2x80x9d In: Proceedings of the Warmwater Fish Culture Workshop, Special Publication No. 3, World Mariculture Society, Charleston, S.C.; Torrissen, O. J. (1986) Aquaculture 53: 271-278; Torrissen., O. J., et al., (1987) xe2x80x9cPigmentation of salmonids-carotenoid deposition and metabolism.xe2x80x9d Northwest and Alaska Fisheries Center). Pigmentation has been achieved using arctic krill as a dietary supplement (Arai, S., et al., (1987) Aquaculture 66: 255-264), but this is expensive.
Canthaxanthin is also used as a food colorant, principally in pink grapefruit juice cocktail mixtures and in poultry, eggs, and aquacultured fish, where it is introduced through the feeds. There are no industrial microbial processes for the production of canthaxanthin, although it is found in the fungus that provides its name, Cantharellus cinnabarinns. 
A recent study estimates that twenty percent of the carotene and xanthophyll supplement markets are dedicated to the natural form as opposed to synthetic materials (Taylor, R. F., and Little. A. D. (1990) xe2x80x9cCarotenoids: products, applications, and markets.xe2x80x9d SPECTRUM Food Industry, pp. 1-11). The principal source of natural microbial xcex2carotene has been photoautotrophic microalgae Dunaliella salinas and Dunaliella bardawil (Ben-Amotz ET AL., (1990) Tibtech 76(5): 121-126; Ben-Amotz, A. (1986). xe2x80x9cxcex2-carotene enhancement and its role in protecting Dunaliella bardawil against injury by high irradiance.xe2x80x9d In: Algal Biomass Technologies, W. R. Barclay and R. P. McIntosh (eds.), J. Cramer, Berlin, pp. 132-147). Approximately one-third of the natural xcex2-carotene produced by Dunaliella is the 9-cis isomer. The differentiation between the 9-cis and all-trans form of xcex2-carotene has been claimed to be nutritionally important (Ben-Amotz et al., (1989) J. Nutrition 119: 1013-1019). A commercial obstacle to using Dunaliella as a source for xcex2-carotene is the difficulty of extracting the pigments from the organisms. This difficulty in extraction correlates with a difficulty in bioexpression of the pigments (defined as the amount of pigment absorbed as a percentage of the amount consumed) when the Dunaliella organism is fed to an animal as a pigment source (Nonomura (1987) U.S. Pat. No. 4,680,314).
A recent comprehensive review (Johnson, E. A., and An, G-W. (1991) Critical Reviews in Biotechnology 11(4): 297-326) cites only two microbial species that are producers of astaxanthin. One is the green microalga Haematococcus, and the other is the yeast Phaffia rhodozyma. Production using Haematococcus has been attempted in photo-autotrophic cultivation in open, fresh-water ponds. Unlike Dunaliella, Haematococcus cannot grow in highly-saline culture conditions, and the fresh-water ponds contaminate easily. Otherwise, production is in closed systems and is very costly.
Astaxanthin is also produced in the Phaffia yeast (An, et al., (1990) Applied and Environmental Microbiology, 55: 116-124; An, et al.(1991) Bio/Technology 9: 70-73). One difficulty that has limited the appeal of both Haematococcus and Phaffia as sources of astaxanthin is very low bioavailability and bioexpression of the astaxanthin in the intact organism, which is attributed to the strong cell walls.
The Thraustochytriales are a relatively obscure order of unicellular organisms rarely described in biology textbooks (Bahnweg, G., and Jackle, I. (1986) xe2x80x9cA new approach to taxonomy of the Thraustochytriales and Lybrinthulales.xe2x80x9d In: The Biology of Marine Fungi, S. T. Moss (ed.), Cambridge University Press, London, pp. 131-140). Thraustochytriales are saprobs, feeding on plant detritus, and are common in marine and estuarine waters, growing naturally at a variety of salinities. Thraustochytriales are known to occur on marine macroalgae as well, and they are found in environments stretching from tropical waters to arctic and Antarctic environments. Reproduction is vegetative or involves the formation of zoospores, which escape through a variety of cleavage mechanisms to produce new sporangia.
Thraustochytriales are described as xe2x80x9ceucarpic and monocentric, with an endobiotic rhizoidal system resembling chytrids but producing planonts like those of oomycetous fungixe2x80x9d (Ninet, L., and Renaut, O. (1975) xe2x80x9cCarotenoids.xe2x80x9d In: The Mycetozoans Olive, L. S. (ed.), Academic Press, NewYork, pp. 529-545). The cell wall is composed of overlapping scales, and their sensitivity to environmental perturbation may well stem from this feature (Bartnicki-Garcia, 1988). Most of the research on these organisms has focused on taxonomic definition. (Emerson, R. (1950) Ann. Rev. Micro. 4: 169-200; Goldstein, S. (1965) Am. J. Bot. 50: 271-279; Margulis, 1970; Margulis et al., (1985) BioSystems 18: 141-147; Moss, S; (1986) xe2x80x9cBiology and phylogeny of the Labrinthulales and Thraustochytriales.xe2x80x9d In: The Biology of Marine Fungi, S. T. Moss (ed.), Cambridge University Press, London, pp. 105-130; Perkins, F. O. (1974) Veroff. Inst. Meeresforsch. Bremen, Suppl. 5: 45-63; Perkins, F. O. (1976) xe2x80x9cFine structure of lower marine and estuarine fungi.xe2x80x9d In: Recent Advances in Marine Mycology, E. B. Gareth Jones (ed.), Elek Science, pp. 279-312; Ragan, M. A., and Chapman, D. J. (1978) A Biochemical Phylogeny of the Protists, Academic Press, New York, pp. 1-5, 147-168; Sparrow, F. K. (1960) Aquatic Phycomycetes. University of Michigan Press, Ann Arbor, pp. 37-38). This includes studies that employ chemical taxonomy based on fatty acid distributions (Ellenbogen et al., (1969) Comp. Biochem. Physiol. 29: 805-811; Findlay, R. H., et al. (1986) xe2x80x9cBiochemical indicators of the role of fungi and Thraustochytrids in mangrove detrital systems.xe2x80x9d In: The Biology of Marine Fungi, S. T. Moss (ed.), Cambridge University Press, London, pp. 91-105). Aside from these fatty acid profiles, no biochemical characterization has been made. There is no literature report of the analysis of specific pigments, although there have been anecdotal reports of yellow-to-orange coloring of the ectoplasmic nets and speculation that some orange or red-colored species contain xanthophyll pigments (Barclay (1992), U.S. Pat. No. 5,130,242).
The present invention relates to a method for producing one or more carotenoid, xanthophyll, or apo-carotenoid pigments. The method comprises providing a microorganism from the Order Thraustochytriales capable of producing an effective amount of the desired pigment, culturing the microorganism under conditions of heterotrophic growth appropriate for effective production of the pigment, and then isolating the pigment from the cultured microorganism. Microorganisms put forth as suitable for this method are of the genera Thraustochytrium and Schizochytrium. Preferred culture conditions and medium are described. Examples are given of production of specific pigments, such xcex2-carotene, lutein, adonirubin, canthaxanthin, and astaxanthin. The present invention also relates to a food product, comprising food material and a microorganism from the Order Thraustochytriales, the microorganism having produced internally an effective amount of one or more carotenoid, xanthophyll, or apo-carotenoid pigments. The food product is used in a method for delivering one or more pigments produced by the microorganism to an animal or human, by feeding the food product to the animal or human.